Fluorescent dyes, also referred to herein as fluorescent compounds, fluorophores and fluorochromes, have many useful applications. For example, fluorescence can be used as a non-destructive way of tracking or analysing many diverse molecules. Such molecules are labelled with a fluorescent dye, enabling the fluorescence of the dye to be detected.
In recent years there has been growing interest in visible red and near-infrared (NIR) fluorescent dyes (>700 nm) for qualitative and quantitative assays. Their simplicity of use, coupled with high sensitivity also ensures a continual expansion of their use for in vitro and in vivo imaging. In terms of commercial application, high sensitivity NIR fluorescence imaging has become an indispensable tool for probing the molecular processes of biological systems in living cells. Its application to non-invasive in vivo animal and human imaging is currently a re-emerging field with applications varying from vascular mapping to tumor diagnosis. NIR optical imaging of tissues is an inexpensive, real-time and non-invasive technique that does not require the use of radionuclides. Recently developed ultra sensitive low noise charge coupled cameras, mathematical models of photon propagation in tissue, and more target-specific molecular probes, have created exciting possibilities in this field. The inherent advantages of NIR fluorochromes over those that absorb in the shorter blue and green wavelengths lie in the dramatic reduction in background autofluorescence which leads to greatly improved sensitivity, increased light penetration of biological tissue, and minimal damaging of the cells/tissue under observation. As an example, in the case of in vivo imaging, it is strongly preferential to use fluorophores with absorption/emission profiles in the far visible red or near infrared spectral (NIR) regions (700 nm-1400 nm), as at lower wavelength, strong interference from endogenous chromophores is problematic.
Despite the optical benefits, there is a surprising scarcity of NIR compounds which have the desired absorption and emission properties. Among the fluorescent platforms used for the construction of visible red/NIR fluorescent probes for bioconjugation, the cyanine dyes are by far the most widely utilised. To date, of the cyanine dyes, only indocyanine green (ICG) has been approved for clinical use. Furthermore, despite their widespread use, the cyanine dyes have disadvantages, including poor photostability and lengthy synthetic routes.
The boron chelated tetraarylazadipyrromethene class of compounds has been found to be relatively easily synthesised and to exhibit excellent spectral properties. Their strong absorption and emissions within the NIR spectral region, together with high photostability make them promising candidates for biological imaging applications. For example, the compound 1 has an absorption λmax at 696 nm and emission at 727 nm (φF=0.36) in formulated aqueous solutions, and absorption λmax at 688 nm (ε=85,000 dm−3 mol−1 cm−1) and emission at 716 nm (φF=0.36) in chloroform.

These promising photophysical characteristics have encouraged the adaption of this class to specific functions such as photodynamic therapy. WO 03/080627 discloses the use of these compounds 1 as photodynamic therapeutic agents. However, such compounds, although useful, also have disadvantages. For example, their commercial application has been limited to date by their inability to be bound to a molecule or particle of interest for tracking or analysing the molecule. Specifically, it is not feasible to add a conjugation group onto the carbon of the methoxy groups of the compound. For the above reasons, a distinct need exists for novel NIR fluorescent dyes.
It is an object of the invention to avoid or mitigate the problems associated with known NIR fluorescent dyes.
It is a further object of the invention to provide novel NIR fluorescent dyes that are stable and are capable of binding to a molecule or particle of interest.
It is a still further object of the invention to provide novel NIR fluorescent dyes that are water-soluble.
It is a still further object of the invention to provide novel NIR fluorescent dyes that have a predictable fluorescence intensity response to environmental changes, for example pH.